The technology of recombinant DNA manipulation and insertion has evolved to the point where it is now possible to genetically engineer many crop plants. In the genetic engineering of crop plants, an exogenous genetic construction is inserted into the genomic DNA of the target plant species. The transformed plants can then express the exogenous gene product encoded by the genetic construction. If germ-line cells are transformed, the plants produced through such a process carry in their genomic DNA the inserted foreign genetic construction, which can thereafter be passed on to the progeny of the plant by normal plant breeding techniques. Using such techniques, it has become commonplace to genetically engineer model species, such as tobacco, petunia, carrot, potato and poplar. The techniques of genetic engineering have recently been extended to the important crop species cotton and soybean. Progress in plant genetic engineering has thus varied from crop plant family to family and the transformation of many species and families is yet to be demonstrated.
The most common technique utilized to transfer foreign genetic materials into plant cells makes use of the common soil-dwelling bacterium, Agrobacterium tumefaciens. A tumefaciens is a plant pathogen that natively harbors a plasmid, referred to as the Ti (tumor-inducing) plasmid, which has the inherent ability to transfer a portion of its DNA (T-DNA) into a target plant cell. By suitable manipulation of the Ti plasmids of Agrobacterium tumefaciens, it is possible to insert a foreign genetic construction into the T-DNA of the Ti plasmid, which is then transformed into susceptible plant cells in tissue culture.
One difficulty in the utilization of Agrobacterium-mediated techniques for perennial fruit plant transformation is that Agrobacterium is very dependent on a species specific interaction between Agrobacterium and the cells of the target species plant. Another difficulty is that not all plant species can yet be regenerated from tissue cultures transformed by Agrobacterium. Cranberry, for example, has not been successfully transformed by Agrobacterium. Other perennial fruits, such as Rubus, have been transformed with Agrobacterium, Graham, J. et al., Plant Cell Tissue Organ Culture 20: 35-39 (1990).
Other techniques for transforming individual cells or cells in tissue culture include direct DNA injection and electroporation of plant protoplast cells. One especially promising alternative method for genetically engineering whole plants involves the coating of DNA or RNA onto small particles which are then physically accelerated into the cells of the target plant tissues, Klein et al., Proc. Natl. Acad. Sci. U.S.A., 88: 8502-8505 (1988). A technique for germline transformation of soybean by particle-mediated transformation has been published, McCabe et al., Bio/Technology, 6: 923-926 (1988).
Genetic transformation of perennial fruits would permit expression of beneficial genes, such as the Bacillus thuringiensis crystal protein (B.t.) gene, which could potentially confer lepidopteran insect resistance. Vaeck, et al., (Nature 238: 33-37 (1987)) discuss insect-resistant plants that had been transformed with the B.t. gene. This toxin has been previously found to be specific to Lepidopteran insects, i.e., the larvae of moths and butterflies. Since caterpillars are a consumer of perennial fruits, the creation of plants having resistance to attack by Lepidopteran larvae would be of significant value.
Cranberry is a good example of a perennial fruit plant that could be beneficially genetically engineered. The American cranberry, Vaccinium macrocarpon Ait., is a woody, low-growing perennial vine. It is native to North America and found growing in temperate lowland marsh areas where the soil is acidic and high in organic matter. Cranberries are cultivated for their tart berries, which are primarily used for juice and sauce products. Cranberries, like most perennial fruits, are asexually propagated.
Cranberry marsh productivity has increased steadily over the years. Improvements in cultural practices have included the use of integrated pest management. However, problems such as insect and weed control are still prevalent, and control of these pests has been estimated to make up 45% of the growers, direct field costs. Currently, substantial amounts of carbamate and organophosphate insecticides are used throughout the growing season for insect control (Mahr et. al., Cranberry Pest Control in Wisconsin, University of Wisconsin--Extension Service Bulletin #A3276, p18, (1988)). Use of these insecticides poses problems of effectiveness and toxicity.
What is lacking in the art is an efficient, economical and rapid method of transforming cranberry and other perennial fruit plants capable of micropropagation and capable of adventitious budding on micropropagated tissue.